Clottable fibrinogen free factor VIII and albumin product and process

ABSTRACT

Lyophilizable, clottable fibrinogen free, Factor VIII is produced on a commercial scale from a fibrinogen containing Factor VIII concentrate by sequentially, precipitating a major portion of the clottable fibrinogen by the addition of an ethylene oxide-polyoxypropylene block copolymer, clotting the remainder of the clottable fibrinogen by the addition of a small amount of a thrombin mimetic enzyme which does not decrease the potency of the Factor VIII, and adding human albumin to the resultant clottable fibrinogen free Factor VIII to stabilize its potency during lyophilization.

This invention relates to the preparation of a Factor VIII product whichis free of clottable fibrinogen. Factor VIII is one of the traceproteins which is required in the clotting of human blood. When FactorVIII is missing from a patient's blood or is for some reasonbiologically inactive, normal clotting does not take place. Onetreatment for such a patient is to add normal Factor VIII to thepatient's blood by injection. A problem arises in this regard becausepreviously it was not feasible on a commerical scale to separate FactorVIII and fibrinogen. Thus, previously a highly potent commerciallyavailable Factor VIII concentrate would also inevitably contain asignificant concentration of fibrinogen. The injection of an excessiveamount of fibrinogen often resulted in kidney failure. For this reasonit was often impossible to administer an effective dosage of Factor VIIIto a patient who needed this material for normal clotting.

According to the present invention, a Factor VIII concentrate isprepared which is free of clottable fibrinogen. The procedures aresuitable for the production of high yields of Factor VIII concentrate ona commercial scale. The product is suitable for injection intohemophiliac patients. Also, this clottable fibrinogen free Factor VIIIconcentrate may, if desired, be lyophilized for purposes of storage andtransportation.

In general the present invention includes a procedure whereby asubstantial proportion of the clottable fibrinogen (more thanapproximately 90 percent) is precipitated in an initial separation stepwith the Factor VIII remaining in solution. The precipitated fibrinogenis separated and discarded. The resulting fibrinogen lean supernate isconcentrated by precipitation and reconstitution to reduce the volume ofliquid. The reconstituted Factor VIII-fibrinogen precipitate is treatedwith a small amount of a thrombin mimetic enzyme which causes theremaining clottable fibrinogen to clot but does not significantly impairthe biological activity of the Factor VIII. These thrombin mimeticenzymes occur naturally in various snake venoms and are isolated andpurified for use in the present invention. The Factor VIII remains inthe supernate and the clotted fibrinogen is discarded. The Factor VIIIwhich is now free of clottable fibrinogen is again precipitated and thesupernate is discarded. The resulting Factor VIII precipitate isredissolved in a small amount of saline solution, and human albumin isadded to stabilize the Factor VIII during lyophilization. The yield ofFactor VIII is as high as approximately 90 percent, and the lyophilizedproduct upon reconstitution exhibits substantially all of the FactorVIII bilolgical activity that it possessed prior to lyophilizaton.

The entire procedure except for lyophilization may be conducted at roomtemperature and preferably at temperatures ranging from about 15 ° to30° centigrade, although temperatures as low as 10° centigrade and ashigh as 35° centigrade may be used. Temperatures below about 10°centigrade tend to impair the potency of the Factor VIII, andtemperatures in excess of about 35 degress centigrade impair thebiological activity of the Factor VIII. At a temperature of about 40°centigrade the potency of the Factor VIII is completely destroyed.

In general, the salt concentration of the Factor VIII solutions whichcontain the Factor VIII are adjusted to physiological values (0.15 molarsodium chloride), although salt concentrations somewhat above and belowphysiological values may be used if desired. After the Factor VIII hasbeen separated from the fibrinogen, it is somewhat sensitive to saltconcentrations so that salt concentrations of about 0.05 to 0.25 molarsodium chloride are preferred for use with Factor VIII containingsolutions after the fibrinogen has been removed. Prior to the removal offibrinogen, salt concentrations of from about 0.05 to 0.50 molar sodiumchloride may be employed if desired without impairing the potency of theFactor VIII or the thrombin mimetic enzyme, and without precipitatingthe Factor VIII from the liquid phase. Sodium citrate may be utilizedthroughout the precedure and in the final lyophilized product if desiredin addition to sodium chloride.

In general the pH of the solutions utilized in the preparation of theclottable fibrinogen Factor VIII product may range from about 6.0 to 8.0and preferably from about 6.5 to 7.0. Below a pH of 6 the fibrinogenforms clots which carry the Factor VIII with them, and above a pH ofabout 8 the yields are so low that substantially no Factor VIII isrecovered. After the fibrinogen has been separated from the Factor VIII,it becomes somewhat more sensitive to pH, and pH values should bemaintained in the range of from about 6.7 to 7.0.

The initial precipitation of a substantial portion of the fibrinongen aswell as the subsequent precipitations of Factor VIII containingmaterials is conveniently accomplished by utilizing differentconcentrations of certain block copolymers. The particular blockcopolymers that are utilized in these selective precipitation proceduresare conveniently prepared by condensing ethylene oxide withpolyoxypropylene polymer. The resultant condensation products are wellknown materials which can be represented by the following structuralformula: ##STR1## Block copolymers which are suitable for these purposesare available from Wyandotte Chemicals Corp. under the designations"Pluronic F-38" and "Pluronic F-68." The Pluronic F-38 material containsabout 80 percent polyoxyethylene units and the polyoxypropylene polymerportion of the molecule has a molecular weight of about 950. Thecondensation product has a molecular weight of about 4,750. The PluronicF-68 material also contains about 80 percent polyoxyethylene units andthe polyoxypropylene polymer portion of the molecule has a molecularweight of about 1,750 with the total molecular weight of thecondensation product being about 8,750. In general these blockcopolymers contain at least about 50 percent ethylene oxide in themolecule, and the polyoxypropylene polymer portion of the molecule has amolecular weight of at least about 900. The block copolymers must bewater soluble at concentrations of at least about 20 percent on a weightper volume basis at temperatures of from about 10° to 35° centigrade.These materials must be nontoxic.

In general the initial separation step in which a substantial portion ofthe fibrinogen is separated by precipitation from a Factor VIIIcontaining supernate is accomplished at a concentration of from about 1to 4.5 gram percent (grams per 100 cubic centimeters), and preferably 3to 4 gram percent of the block copolymer at a salt concentration ofabout 0.15 molar sodium chloride and a temperature of from about 15° to30° centigrade. At concentrations below about 1 gram percent theprocedure does not remove a sufficient amount of fibrinogen, and atconcentrations above about 4.5 gram percent a substantial portion of theFactor VIII is also precipitated. The partial separation of fibrinogenand Factor VIII using Pluronic F-38 and F-68 is suggested in Fekete etal U.S. Pat. No. 3,770,631.

In subsequent steps where it is desired to precipitate all of the FactorVIII the concentration of the block copolymer ranges from about 6 to 10gram percent, and preferably from about 8 to 10 gram percent. Below 6gram percent block copolymer the yield of Factor VIII is very low. Aboveabout 10 gram percent substantially all of the bacteria which may bepresent are precipitated. As the temperataure is decreased below about15 degrees centigrade the concentration of the block copolymer should bereduced slightly so as to avoid the precipitation of excessive amountsof bacteria which may be present in blood plasma fractions.

The thrombin mimetic enzymes or regents are used to clot and thusseparate the residual fibrinogen which remains after the initialfibrinogen separation step. These reagents, unlike thrombin, do notimpair the biological activity of Factor VIII. These thrombin mimeticreagents are extracted from the venom of various snakes including, forexample, the Malayan pit-viper (Agkistrodon rhodostoma), the SouthAmerican snake (Bothrops atrox), and the snakes (Crotalus terrificus)and (Crotalus adamanteus). These thrombin mimetic enzymes are availableunder the brand names "Arvin" (Twyford Laboratories, London, England),"Ancord" (Abbott Laboratories, North Chicago, Illinois), and"Reptillase" (Abbott Laboratories, North Chicago, Illinois). It hadpreviously been suggested that these thrombin mimetic enzymes might beused to prepare low yields of Factor VIII which is free of fibrinogen,Greed D.: "A Simple Method for the Purification of Factor VIII(Antihemophiliac Factor) Employing Snake Venom." J. Lab. Clin. Med. 77:153-158 (1971).

The respective fibrinogen and Factor VIII precipitation steps, otherthan the clotting step, may be carried out using precipitation reagentsother than the ethylene oxide-polyoxypropylene block copolymercondensation products; for example, polyethylene glycol may be used withsubstantially diminished yields of Factor VIII, as taught, for example,by A. Polson and C. Ruiz-Bravo: "Fractionation of Plasma withPolyethylene Glycol" Vox Sang. 23: 107-118 (1972). Ethanol, ammoniumsulfate, and glycine require the use of low temperatures which aredestructive to the potency of highly concentrated Factor VIII. Dialysisprocedures may also be used if desired, although they are not generallyeconomically feasible. The block copolymers produce the best results andare preferred. Polyethylene glycol produces acceptable results for acommercial scale operation.

The potency of the thrombin mimetic enzymes is given herein in terms ofthrombin units with one Iowa unit of thrombin being equal to one unit ofthrombin mimetic enzyme. One Iowa unit of thrombin is that amount ofthrombin which will clot standard fibrinogen in 15 seconds at 28°centigrade. The biological activity or potency of Factor VIII ismeasured in units of Factor VIII with one unit of Factor VIII being thatamount of Factor VIII which occurs in one milliliter of freshly drawnpooled plasma. The Factor VIII is measured in units as is the thrombinmimetic enzyme, because this protein is present in such minutequantities that its weight and volume cannot be detected withconventional laboratory equipment.

The concentration of thrombin mimetic enzyme which is utilized in thepreparation of clottable fibrinogen free Factor VIII should be kept aslow as possible. In high concentrations, the enzyme attacks the FactorVIII and also tends to be carried over into the final product. Theenzyme if injected into a patient will cause excessive bleeding, eventhough it causes the clotting of fibrinogen in vitro. Thus, theinjection of the enzyme into a hemophiliac patient is very undesirable.By removing a substantial portion of the fibrinogen before the thrombinmimetic enzyme is used and concentrating the resultant fibrinogen leanmaterial, it is possible to utilize enzyme concentrations which do notsignificantly impair the potency of the Factor VIII. The action of theenzyme appears to be catalytic in nature so that it is effective even invery minute concentrations. The concentrations should, however, beadequate to cause the clotting of all of the clottable fibrinogen in theadmixture within approximately 24 hours. Lower concentrations of theenzyme will result in complete clotting of the fibrinogen but severaldays will be requied to accomplish this. The Factor VIII tends to loseits biological activity if it remains in solution for too long a periodof time. For this reason extremely low concentrations of thrombinmimetic enzyme do not produce satisfactory yields of Factor VIII. Also,extended clotting times permit the growth of undesired bacteria in theadmixture. Concentrations of thrombin mimetic enzyme which are effectivein clotting the fibrinogen within approximately 24 hours atapproximately 22° centigrade without significantly impairing the potencyof the Factor VIII are from about 0.5 units of enzyme to 3 units ofenzyme per 1,000 milligrams of fibrinogen, and preferably from about0.75 units of enzyme to 2 units of enzyme per 1,000 milligrams offibrinogen. At a concentration of 0.15 units of enzyme to 1,000milligrams of fibrinogen clotting requires approximately 40 hours, andat a concentration of 3 units of enzyme per 1,000 milligrams offibrinogen the yield of Factor VIII drops to approximately 50 percent.

In general, the concentration of clottable fibrinogen should be reducedto less than approximately 0.2 milligrams of clottable fibrinogen perunit of Factor VIII before the thrombin mimetic enzyme is used to clotthe remaining clotting fibrinogen. At higher concentrations offibrinogen excessive amounts of thrombin mimetic enzyme relative to theFactor VIII are required to accomplish clotting within the desired timeand the potency of the Factor VIII is impaired. Preferably the clottablefibrinogen lean Factor VIII concentrate contains less than about 0.15milligrams of fibrinogen per unit of Factor VIII. Fresh pooled plasmacontains about 2 milligrams of fibrinogen per unit of Factor VIII.

After the initial fibrinogen removal step, the clottable fibrinogen leanFactor VIII aqueous admixture is concentrated to a Factor VIII potencyof at least approximately 15 and preferably at least about 35 units permilliliter so as to avoid excessive dilution of the thrombin mimeticenzyme.

Factor VIII tends to lose its potency if it is stored and transported inthe liquid form. Accordingly, it is necessary to lyophilize the FactorVIII for purposes of transportation and storage. Prior to the presentinvention it had generally been believed that it was not possible tolyophilize clottable fibrinogen free Factor VIII without substantiallydestroying its potency. According to the present invention, the additionof human albumin to the clottable fibrinogen free Factor VIII materialwill stabilize the Factor VIII so that it may be lyophilized andreconstituted without losing any significant amount of its potency. Atleast about 0.5 gram percent of human albumin (grams per 100milliliters) is required to stabilize the Factor VIII duringlyophilization. Preferably, the concentration of human albumin in theclottable fibrinogen free Factor VIII concentrate solution is at leastone gram percent. Greater quantities of human albumin may be used up tothe limits of the solubility of this material; however, no advantagesare gained by increasing the quantity of human albumin, and theincreased quantities tend to render the product less suitable forinjection into hemophiliac patients. The lyophilized product generallycontains at least about 10 and preferably at least about 20 units ofFactor VIII per milligram of human albumin.

The following examples are submitted to illustrate and not to limit theinvention.

EXAMPLE I

The purpose of this example is to illustrate the preparation of highpotency fibrinogen free Factor VIII concentrate by a procedure which issuited for use on a commercial scale.

A crude fibrinogen containing liquid Factor VIII concentrate wasselected as the starting material. This Factor VIII concentrate had apotency of 4.7 units per milliliter of Factor VIII, as determined by invitro bioassay procedures. The 266 milliliter starting sample of thisFactor VIII concentrate contained 2,700 milligrams of fibrinogen per 100milliliters of sample. The total weight of fibrinogen in the 266milliliter starting sample was 7,182 milligrams, as determined byconventional in vitro bioassay procedures.

INITIAL FIBRINOGEN PRECIPITATION

The pH of this 266 milliliter sample was adjusted to a value of 6.5, andthe temperature was maintained at about 22 degrees centigrade.

About 3.5 percent on a weight per volume basis (grams per 100 cubiccentimeters) of a block copolymer was added to the sample of crudeFactor VIII concentrate. The block copolymer was an ethyleneoxide-propylene glycol condensation product in which about 80 percent ofthe block copolymer consisted of polyoxyethylene units. Thepolyoxypropylene portion of the polymer had a molecular weight of about1,750. The total molecular weight of the copolymer was about 8,750. Thismaterial is available commercially from Wyandotte Chemicals Corp. underthe designation "Pluronic F-68." The resulting admixture containing thedissolved block copolymer was stirred for about 20 minutes during whichperiod of time a precipitate formed. The precipitate was centrifugeddown at 5,000 gravities for a period of about 15 minutes. The FactorVIII containing supernate was recovered, and the precipitate wasdiscarded. The precipitate consisted primarily of fibrinogen. Samples ofthe resulting fibrinogen lean retained supernate were withdrawn andanalyzed by in vitro bioassay procedures for Factor VIII potency andfibrinogen concentration. The Factor VIII potency was found to be about4.8 units per milliliter. The supernate contained about 200 milligramsof fibrinogen. The volume of the supernate remaining after the sampleshad been withdrawn for assay purposes was 234 milliliters.

INITIAL FACTOR VIII PRECIPITATION

The pH of this 234 milliliter sample was adjusted to a value of about6.88. The temperature of this sample was about 22° centigrade. An amountof the same block copolymer utilized previously (Pluronic F-68)sufficient to bring the total concentration of block copolymer to 10percent on a weight per volume basis (grams per 100 cubic centimeters)was added to the mixture. The admixture was stirred for about 15 minutesduring which period of time a precipitate formed therein. The FactorVIII containing precipitate was centrifuged down for about 15 minutes at5,000 gravities. Substantially all of the Factor VIII and fibrinogenwere in this precipitate. The supernate was discarded. The saltconcentration of the crude fibrinogen containing Factor VIII samplewhich was used as the starting material in this example was at thephysiological value of 0.15 molar sodium chloride. This saltconcentration remained at about this value in the liquid phasethroughout the procedure to this point. The precipitate recovered fromthis step was very rich in Factor VIII.

REDISSOLVED REDUCED FACTOR VIII

This Factor VIII rich precipitate was redissolved in citrated saline (1part by volume of 0.1 molar sodium citrate and 4 parts by volume of 0.15molar sodium chloride solution). The volume of the redissolved reducedFactor VIII rich precipitate was 30 milliliters. The pH was adjusted toa value of 6.88 and the temperature was maintained at a value of about22° centigrade. The Factor VIII potency, as determined by conventionalin vitro bioassay procedures, was about 50 units per milliliter. The 30milliliters of redissolved reduced precipitate contained about 200milligrams of fibrinogen.

ENZYMATIC SEPARATION OF FIBRINOGEN

The remaining fibrinogen was separated from the redissolved reducedprecipitate by adding 0.2 units of a thrombin mimetic enzyme to theredissolved reduced precipitate. The concentration of enzyme was about 1unit of enzyme to 1,000 milligrams of fibrinogen. The thrombin mimeticenzyme was a purified enzyme fraction of Malayan pit-viper venom whichis available under the brand name "Ancrod" from Abbott Laboratories.This enzyme does not activate Factor VIII so that the Factor VIIIretained its potency. A solid mass (clot) formed in the resultantadmixture in about 24 hours. The resultant mass was centrifuged at 5,000gravities for about 20 minutes, and the Factor VIII rich supernate wasrecovered. All of the clottable fibrinogen was removed in the solidmass. Some potency of Factor VIII was lost in this step, but the losswas less than approximately 10 percent and was not considered to besignificant.

FIBRINOGEN FREE FACTOR VIII PRECIPITATE

The pH of the Factor VIII rich fibrinogen free concentrate was checkedto insure that it remained at a value of 6.88. The above described blockcopolymer (Pluronic F-68) was added to the fibrinogen free supermate.The quantity of block copolymer was sufficient to bring theconcentration of block copolymer in the supernate to a value of 10percent on a weight per volume basis (grams per 100 cubic centimeters).The admixture containing the dissolved block copolymer was stirred forabout 20 minutes at a pH of 6.88 and a temperature of about 22°centigrade. A Factor VIII containing precipitate was formed which wasseparated from the supernate by centrifuging the admixture at 5,000gravities for about 20 minutes. The supernate was discarded.

FACTOR VIII REDISSOLVED

The resulting Factor VIII precipitate was redissolved in one milliliterof citrated saline having the composition described above and theresultant solution was further diluted for assay purposes to a totalvolume of 8 milliliters. In order to maintain the potency of the FactorVIII during the lyophilization 1 weight percent (grams per 100 cubiccentimeters) of human albumin was added. This 8 milliliter volume ofstabilized Factor VIII concentrate contained no clottable fibrinogen, asdetermined by conventional in vitro bioassay procedures.

YIELD

The crude fibrinogen containing Factor VIII concentrate startingmaterial contained 1,250 units of Factor VIII, and the finalconcentrated fibrinogen free product contained 975 units of Factor VIIIfor a yield of 78 percent. A portion of the Factor VIII was lost in thebioassay procedures which were carried out at various points in thepreparation of the fibrinogen free highly potent Factor VIIIconcentrate. Disregarding loses due to the withdrawal of samples forbioassay purposes, the yield is approximately 90 percent. A yield ofthis magnitude in a commercial scale operation results in theconservation of raw material which must be obtained from human donorsand is in short supply. In a commercial scale operation the startingmaterial would have a volume of at least 10 liters with a potency ofapproximately 4.7 units per milliliter of Factor VIII.

LYOPHILIZATON FOR STORAGE AND TRANSPORTATION

The salt concentration was adjusted to 0.15 molar sodium chloride andthe product was lyophilized. Upon reconstitution the lyophilized productwas found to have retained its potency and to be suitable foradministration to hemophiliac patients. No trace of the thrombin mimeticenzyme remained in the product. The lyophilization procedure requiredabout 36 hours. The balance of the procedure, exclusive of thelyophilization procedure, required about 32 hours. Except for thelyophilization step the procedure was carried out at room temperature.

EXAMPLE II

Repetition of Example I twice utilizing sodium chloride concentrationsthroughout of 0.25 and 0.05 molar sodium chloride, respectively, resultsin achieving satisfactory yields of Factor VIII.

Repetition of Example I utilizing the lower molecular weight blockcopolymer which is identified by the brand name "Pluronic F-38," anddescribed hereinabove, produces satisfactory yields. In general,concentrations of the block copolymer are about 0.5 gram percent higherthan for the Pluronic F-68, so that the initial fibrinogen precipitationstep utilizes a concentration of 4 gram percent, and the Factor VIIIprecipitation steps utilize a concentration of about 12 gram percent.

Repetition of the lyophilization procedures of Example I results in thepreparation of a lyophilized material, which when placed in a nonemilliliter capacity vial may be reconstituted with one milliliter ofsterile water so as to produce an injectable Factor VIII concentratematerial. The lyophilization procedure is repeated on successive sampleshaving different Factor VIII potencies so that the reconstituted onemilliliter solution will contain respectively 300, 1,000 and 2,500 unitsof Factor VIII (30, 100, and 250, respectively, units of Factor VIII permilligram of human albumin). Each of these is suitable for injection inan hemophiliac patient. Because of the absence of fibrinogen, therematerials are suitable for intramuscular injection.

What is claimed is:
 1. Process comprising:selecting an admixturecontaining clottable fibrinogen and Factor VIII; removing most of theclottable fibrinogen from said admixture to produce a clottablefibrinogen lean Factor VIII product; concentrating said product toproduce a concentrate; admixing said concentrate with an amount ofthrombin mimetic reagent sufficient to clot all of the clottablefibrinogen in said concentrate within approximately 24 hours withoutsignificantly impairing the potency of said Factor VIII, and allowingsaid clottable fibrinogen to form a clot; separating said clot andretaining a clottable fibrinogen free Factor VIII concentrate; admixingsaid retained clottable fibrinogen free Factor VIII concentrate with anamount of human albumin sufficient to stabilize said Factor VIII; andlyophilizing the resultant stabilized Factor VIII.
 2. Process of claim 1wherein approximately 90 percent of the clottable fibrinogen is removedfrom said admixture.
 3. Process of claim 1 wherein said clottablefibrinogen lean Factor VIII product is concentrated to a Factor VIIIpotency of at least approximately 15 units.
 4. Process of claim 1wherein said process is carried out at a temperature of from about 10°to 35° centigrade.
 5. Process of claim 1 wherein said process is carriedout at a salt concentration of from about 0.05 to 0.25 molar sodiumchloride.
 6. Process of claim 1 wherein said process is carried out at apH of from about 6.0 to 8.0.
 7. Process of claim 1 wherein saidresultant stabilized Factor VIII is lyophilized to produce a lyophilizedproduct containing at least about 20 units of Factor VIII per milligramof human albumin.
 8. A process of lyophilizing clottable fibrinogen freeFactor VIII concentrate comprising:preparing an aqueous liquid admixtureof said Factor VIII concentrate and at least about 0.5 percent on aweight per volume basis of human albumin; and lyophilizing said aqueousliquid admixture.
 9. A lyophilized clottable fibrinogen free Factor VIIIproduct comprising clottable fibrinogen free Factor VIII and humanalbumin and containing at least about 10 units of Factor VIII permilligram of albumin.
 10. A lyophilized clottable fibrinogen free FactorVIII product comprising clottable fibrinogen free Factor VIII and humanalbumin and containing at least approximately 100 units of Factor VIIIper milligram of albumin.
 11. A lyophilized clottable fibrinogen freeFactor VIII product comprising clottable fibrinogen free Factor VIII andhuman albumin and containing at least approximately 250 units of FactorVIII per milligram of albumin.
 12. A lyophilized clottable fibrinogenfree Factor VIII product comprising clottable fibrinogen free FactorVIII and at least about 0.5 percent on a weight per volume basis ofhuman albumin.
 13. Process comprising:admixing a clottable fibrinogenlean Factor VIII concentrate containing less than approximately 0.2milligrams of clottable fibrinogen per unit of Factor VIII with fromabout 0.5 to 3 units per 1000 milligrams of clottable fibrinogen of athrombin mimetic reagent that does not significantly impair the potencyof said Factor VIII, and allowing the clottable fibrinogen to form aclot in the resultant admixture separating said clot from said resultantadmixture and recovering a clottable fibrinogen free Factor VIIIconcentrate; admixing said clottable fibrinogen free Factor VIIIconcentrate with at least about 0.5 percent on a weight per volume basisof human albumin to stabilize said Factor VIII; and lyophilizing theresultant stabilized Factor VIII concentrate.